Grassland degradation in the Inner Mongolia grassland became seriously since the end of 20th century, because of the rapid expansion of livestock numbers and the development of economy. Overgrazing in this region is one of the main causes of grassland degradation. In this paper, soil microbial biomass carbon (Cmic) and nitrogen (Nmic) under continuous free-grazing and exclusion of livestock were examined in Nuogusitai (NGST) of Tongliao and Haisilamu (HSLM) of Chifeng in Inner Mongolia. Our results showed that Cmic and Nmic content significantly decreased in grazing grassland compared to the exclosure grassland in both NGST and HSLM. In NGST, the average Cmic (79.2 to 881.1 mg C kg-1) and Nmic (6.1 to 64.8 mg C kg-1) were lower than Cmic (452.7 to 1218.1 mg C kg-1) and Nmic (48.4 to 152.6 mg C kg-1) in HSLM. Furthermore, after grazing Cmic and Nmic decreased 81.39, 69.51% and 75.48, 67.12% in 0-5 and 5-15 cm layers, respectively in NGST. These were all greater than in HSLM which were 42.12, 30.00% and 67.12, 31.40%, respectively. The lower Cmic and Nmic concentration in grazing grassland than in exclosure might due to reduction in organic matter into soil by livestock grazing and destruction of vegetation root by trampling and the lower Cmic in Nmic in NGST than HSLM was mainly the result of higher soil pH and soil substrate. Cmic/Corg ratio in HSLM (3.1 to 4.6) was higher than NGST (0.2 to 0.4). The higher Cmic concentration and Cmic/Corg in HSLM suggest a great potential for carbon sequestration in neutral soil than in alkaline soil. Therefore, to restore grassland and alleviate the grassland degradation, exclosure should be encouraged in semi-arid grassland.